1. Field of the Invention
This invention relates to the field of recombinant DNA technology, especially to nucleic acid sequences useful for constructing a transgenic mammal. More specifically, the invention concerns expression of a transgene in certain tissues or organs of a mammal.
2. Description of Related Art
1. Tissue Specific Expression
Production of a transgenic mammal involves the insertion of a nucleic acid sequence, often called a transgene, which codes for a particular polypeptide, into one or more chromosomes of the mammal. This is typically accomplished by inserting the transgene into the pronucleus of an isolated mammalian egg. The transgene becomes incorporated into the DNA of the developing embryo. This embryo is then implanted into a surrogate host for the duration of gestation. The offspring of the surrogate host are evaluated for the presence of the transgene.
Expression of the transgene, i.e., production of the protein encoded by the transgene nucleic acid sequence, may confer a new phenotype on the mammal. Depending on the transgene(s) inserted into the animal and the level of expression of the transgene in the mammal, the mammal may become more or less susceptible to a particular disease or series of diseases. Such transgenic mammals are valuable for in vivo screening and testing of compounds that may be useful in treating or preventing the disease(s), and/or for developing methods useful in diagnosing the disease.
While methods for insertion of a novel gene into a mammal have developed rapidly, several problems with the application of this technology remain. One such problem concerns limiting expression of the gene primarily to a selected tissue or tissues where expression is desired.
Enhanced and/or specific expression of a gene in a select tissue or tissues of a mammal is complex. Expression of a gene is typically regulated at least in part by a non-coding nucleic acid sequence termed a promoter. The promoter is often located near or adjacent to the nucleic acid sequence encoding the polypeptide to be expressed. Frequently, the activity of a promoter is in turn regulated by other nucleic acid sequences termed enhancers and suppressors (also known as silencers). Enhancers increase the level of expression of the gene while suppressors or silencers decrease expression. The location of enhancers and suppressors along a nucleic acid sequence with respect to the promoter and coding sequence is quite varied for different genes. Enhancers and suppressors may be located near or adjacent to the promoter, i.e., within about 1 kilobase (kb) along a strand of DNA (chromosome or vector), or may be located at a much greater distance, e.g., up to 50 kb or more away from the promoter on a chromosome and still exert an effect on the activity of the promoter. Further, they may be located upstream (i.e., 5' to the promoter and coding sequence), or downstream (3' to the promoter and coding sequence). Such positioning for promoter activity is a function of both the type of promoter and the type of enhancer or suppressor used. To further complicate the regulation, enhancers and suppressors may exert their effect on the promoter of more than one gene within a chromosomal locus.
Several enhancers and suppressors have been identified. For example, the level of expression of the gene encoding transthyretin is affected by an enhancer element located about 2 kb upstream from the promoter (Yan et al., EMBO J., 9:869-AFM8 [1990]). Liver specific expression of the albumin gene is regulated by an enhancer located about 10 kb upstream of its promoter (Hammer et al., Science, 235:53-58 [1987]). Tissue specific regulation of the alpha-fetoprotein gene involves three enhancer elements located 1 to 7 kb upstream of the transcription start site of the gene (Pinkert et al., Genes & Dev., 1:268-276 [1987]).
Another enhancer is the hepatocyte-specific control region, or "HCR". The human HCR is believed to be about 774 base pairs (bp) in size or less (Simonet et al., J. Biol. Chem., 268: 8221-8229 [1993]), but has recently been reported to be at least somewhat active as a 150 to 154 bp fragment (Breslow, Proc. Natl. Acad. Sci. USA, 90:8314-8318 [1993]; Shacter et al., J. Lipid Res., 34:1699-1707 [1993]). The HCR is located on chromosome 19, about 18 kilobases (kb) downstream of the apolipoprotein E (apoE) promoter/gene sequence, about 9 kilobases downstream of the apolipoprotein C-I (apoC-I) promoter/gene sequence, and about 2 kilobases (kb) upstream of the apolipoprotein C-I (apoC-I') pseudogene sequence (Simonet et al., [1993], supra; Simonet et al., J. Biol. Chem., 266:8651-8654 [1991]; Simonet et al., J. Biol. Chem., 265:10809-10812 [1990]; Taylor et al., Current Opinion in Lipidol., 2:73-80 [1991]). The HCR appears to be important in expression of the genes ApoE and ApoC-I in the liver; in its absence, these genes are not expressed at detectable levels in this tissue (Simonet et al. [1993], supra).
The effect of the HCR on a heterologous promoter has been evaluated in transgenic mice. The apolipoprotein A-IV promoter and coding sequence were ligated to a 1.7 kb nucleic acid sequence containing the HCR. Transgenic mice containing this construct had high levels of expression of apolipoprotein A-IV in the liver (Simonet et al., supra).
2. Interleukin-8
The interleukins are a group of naturally occurring proteins that act as chemical mediators of the differentiation processes for red and white blood cells. One of the interleukins, IL-8 (also known as Neutrophil Activating Peptide-1, or NAP-1), has been shown to be a neutrophil chemoattractant with the ability to activate neutrophils and stimulate the respiratory burst (Colditz et al., J. Leukocyte Biol., 48:129-137 [1990]; Leonard et al., J. Invest. Derm., 96:690-694 [1991]). IL-8 has been termed a proinflammatory cytokine due to its involvement in neutrophil recruitment to sites of acute and chronic inflammation.
Zwahlen et al. (Int. Rev. Exp. Path., 34B:22-42 [1993]) describe some effects of IL-8 injected into some rodents. When injected intradermally into rats, IL-8 induced neutrophil infiltration at the site of injection. Intravenous injection of IL-8 into rabbits resulted in neutrophil sequestration in the lungs.
Vogels et al. (Antimicrobial Agents and Chemotherapy, 37:276-280 [1993]) describe the effect of administering IL-8 to mice either before or after infection of the mice with three different pathogens. Under certain conditions, administration of IL-8 was shown to have a detrimental effect on the survival of the mice.
Van Zee et al. (J. Immunol., 148:1746-1752 [1992]) describe administration of IL-8 to baboons. The animals developed neutropenia rapidly after IL-8 administration. This neutropenia is transient and is followed by a marked granulocytosis which persists for as long as IL-8 is present in the circulation.
Burrows et al. (Ann. NY Acad. Sci., 629:422-424 [1991]) show that guinea pigs injected with IL-8 had a higher level of T-lymphocyte and eosinophil accumulation in the lung than did control animals.
3. Keratinocyte Growth Factor
Keratinocyte growth factor (KGF) is a mitogen that has been identified as specific for epithelial cells, especially keratinocytes (Rubin et al., Proc. Natl. Acad. Sci. USA, 86:802-806 [1989]; Finch et al., Science, 245:752-755 [1990]; Marchese et al., J. Cell Physiol., 144:326-332 [1990]). KGF has shown potential for repair of epidermal tissues such as the skin, and epithelial tissues of the digestive tract. The DNA encoding KGF has been cloned and sequenced (PCT 90/08771, published Aug. 9, 1990).
Guo et al. (EMBO J., 12:973-986 [1993]) have prepared a transgenic mouse containing a transgene constructed of the human keratin 14 promoter and the human keratinocyte growth factor gene. The mouse showed a number of phenotypic differences as compared with non-transgenics such as wrinkled skin and reduced hair follicle density.
4. Monocyte Chemoattractant Protein
Monocyte chemoattractant protein (also known as MCP-1) is a protein that is produced by activated leukocytes in response to certain stimuli. The gene encoding human MCP-1 has been cloned and sequenced (Furutani et al., Biochem. Biophys. Res. Comm., 159:249-255 [1989]; Yoshimura et al., Chemotactic Cytokines, Westwood et al., eds. Plenum Press, NY [1991], pp.47-56). MCP-1 serves to attract monocytes to the site of its release, and is believed to be involved in the cellular immune response and in acute tissue injury (Leonard et al., Immunol. Today, 11:97-101 [1990]). MCP-1 has been shown to be produced by some tumor cells in vitro, and in human metastatic melanomas in vivo (Graves et al., Am J. Pathol., 140:9-14 [1992]).
5. Human Afamin
Afamin ("AFM") is a novel protein recently identified in human serum. AFM has a molecular weight of about 87,000 daltons when run on SDS_PAGE, and shares significant homology to members of the albumin family of proteins including vitamin D binding protein (VDB), alpha fetoprotein, and albumin. In addition, AFM has the characteristic pattern of disulfide bonds observed in this family. AFM cDNA has been stably transfected into Chinese hamster ovary cells, and recombinant AFM (rAFM) has been purified from the conditioned culture medium of these cells. Both AFM and rAFM react with a polyclonal antibody that was raised against a synthetic peptide derived from the deduced amino acid sequence of AFM. There is a need in the art to provide in vivo systems for evaluating the effects of one or more genes on certain diseases.
Accordingly, it is an object of this invention to provide a mammal containing a nucleic acid construct comprising a transgene, and expressing the transgene, where the mammal may be used as an in vivo system to analyze the course of a disease.
It is a further objective to provide a transgene nucleic acid construct and an expression vector that enhance tissue specific expression of a transgene in liver tissue of a transgenic mammal.
Other such objects will readily be apparent to one of ordinary skill in the art.